PROJECT SUMMARY Purinergic signaling plays fundamental roles in activities of the nervous system as diverse as neuroprotection, synaptic transmission, nociception, inflammation, and taste. This process is initiated by releasing adenosine triphosphate (ATP) across the membrane through the classic exocytosis or ATP-permeable channels into the synaptic cleft; the ATP then binds downstream receptors on an adjacent cell. The pannexin family is one of the key ATP-permeable channels and consists of three family members, PANX1-3. PANX1 is the best characterized functionally, and it plays crucial roles in a variety of contexts, including blood pressure regulation, glucose uptake, apoptotic cell clearance, and human oocyte development. Although PANX2 and PANX3 have been less studied than PANX1, they are important in neuronal development, ischemia-reperfusion injury, and skin/skeleton development. Thus, the PANX channels have emerged as promising therapeutic targets for a diverse range of diseases. The PANX1-3 are nonselective, large-pore ion channels, and they are predicted to share a four-transmembrane- helix (4-TM) topology with connexins, innexins, and volume-regulated anion channels. Biochemical and physiological studies provide a consensus view that PANX family members form hexameric channels but do not form gap junctions. PANX can be modulated by various factors, including mechanical scratch, extracellular potassium, intracellular calcium, phosphorylation, and caspase-dependent cleavage, but the molecular mechanisms aren?t known. PANX1 activity is modulated by a wide range of small-molecule compounds, but most of them are not specifically targeting PANX1. There is currently no well-characterized agent that modulates the activity of PANX2 and PANX3. Although PANXs are central to human physiology and are potential targets of therapeutic agents, we do not know their structures. We do not understand, in molecular detail, how the channel is activated or inhibited, or how it is modulated by small molecules binding at specific sites. In this proposed work, we will carry out in-depth structural and functional studies of the three pannexin channels to understand how these molecules work. We have determined the first cryo-EM structure of human PANX1 in the apo state at 3.7 and found a heptameric assembly. We have also shown that human PANX1 can be purified in a native-like lipid environment. Building on this preliminary data, we propose to continue the structural studies of these family members, combined with complementary electrophysiology experiments, proteolipsome-based dye transfer assays, binding assays, and other functional approaches, to define the molecular basis for a comprehensive gating mechanism. We will also locate the binding sites of various drugs and the molecular basis underlying their actions on PANX channels, using a combination of structural and functional approaches. These advances will provide a solid foundation for developing new drugs against PANX-linked diseases and for a deeper understanding of the function of the ATP release channel family.